Review pubs.acs.org/CR
SERS-Activated Platforms for Immunoassay: Probes, Encoding Methods, and Applications Zhuyuan Wang, Shenfei Zong, Lei Wu, Dan Zhu, and Yiping Cui* Advanced Photonics Center, Southeast University, Nanjing 210096, Jiangsu, China ABSTRACT: Owing to their excellent multiplexing ability, high sensitivity, and large dynamic range, immunoassays using surface-enhanced Raman scattering (SERS) as the readout signal have found prosperous applications in fields such as disease diagnosis, environmental surveillance, and food safety supervision. Various ever-increasing demands have promoted SERS-based immunoassays from the classical sandwich-type ones to those integrated with fascinating automatic platforms (e.g., test strips and microfluidic chips). As recent years have witnessed impressive progress in SERS immunoassays, we try to comprehensively cover SERS-based immunoassays from their basic working principles to specific applications. Focusing on several basic elements in SERS immunoassays, typical structures of SERS nanoprobes, productive optical spectral encoding strategies, and popular immunoassay platforms are highlighted, followed by their representative biological applications in the last 5 years. Moreover, despite the vast advances achieved to date, SERS immunoassays still suffer from some annoying shortcomings. Thus, proposals on how to improve the SERS immunoassay performance are also discussed, as well as future challenges and perspectives, aiming to give brief and valid guidelines for choosing suitable platforms according to particular applications.
CONTENTS 1. Introduction 1.1. Historical View of Immunoassay Protocols 1.2. SERS-Based Immunoassay (History, Design, and Advantages) 2. Principles of SERS-Based Immunoassay 2.1. Construction of SERS-Based Immunoassays 2.2. Design and Fabrication of SERS Probes 2.2.1. Optical Label: Organic and Inorganic Raman Reporters 2.2.2. Signal Amplifiers: Metal Nanoparticles 2.2.3. Protection Layer: Silica, Polymers, Etc 2.2.4. Targeting Molecules: Antibodies and Aptamers 3. Platforms for SERS-Based Immunoassay 3.1. SERS-Based Immunoassay on Solid Substrate 3.1.1. Nonmetallic Substrate 3.1.2. Metallic Substrate 3.2. SERS-Based Immunoassay in Liquid Phase (Suspension Array) 3.2.1. Nonmagnetic Substrate 3.2.2. Magnetic Substrate 3.3. Immunoassays on a SERS−Microfluidic Chip 3.3.1. Microfluidic Channels 3.3.2. Droplet Microfluidics 3.4. SERS-Based Immunoassay on Optical Fibers 3.5. Paper-Based SERS Platforms 3.6. Hydrophobic SERS Substrates 3.7. Comparison between Different Platforms for SERS-Based Immunoassay © 2017 American Chemical Society
4. SERS Encoding for Multiplex Detection 4.1. Design and Fabrication of SERS Encoders 4.2. SERS Spectral Encoding Strategy 4.2.1. Raman-Frequency-Based Encoding 4.2.2. Signal-Intensity-Based Encoding 4.3. SERS-Included Joint Encoding 4.3.1. SERS−Fluorescence Joint Spectral Encoding 4.3.2. Spectral−Spatial Joint Encoding 5. Applications of SERS-Based Immunoassay 5.1. Analysis of IgGs 5.2. Study of Disease Biomarkers 5.2.1. Detection of Protein Biomarkers 5.2.2. Detection of miRNAs and Circulating DNAs 5.2.3. Detection of Telomerase Activity and Telomere Length Measurement 5.3. Identification of Bacteria and Viruses 5.4. Detection of Ions and Toxins 5.5. Sorting of Cells 5.6. Multiplex Biochemical Analysis Using SERS Encoders 5.6.1. Multiplex Immuno-Detection of Biomolecules and Cells 5.6.2. Multiplex Ion Detection 6. Challenges and Perspectives 6.1. Improving the Reproducibility of SERSBased Immunoassay 6.1.1. Uniformity of the Immune Substrates
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Received: January 12, 2017 Published: May 23, 2017
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Chemical Reviews 6.1.2. Stability of SERS Nanoprobes 6.2. Real Sample Detection 6.2.1. Serum and Plasma 6.2.2. Whole Blood 6.2.3. Other 6.3. Point-of-Care Testing (POCT) 6.4. Practical SERS-Encoding Technique 6.5. Combining SERS with Other Techniques for Multimodal Immunoassay 6.5.1. SERS−SPR Immunoassay 6.5.2. SERS−Fluorescence Immunoassay 6.5.3. SERS−Electrochemistry Immunoassay 6.5.4. Immunoassay with Hyperbolic Metamaterials 7. Conclusion Author Information Corresponding Author ORCID Notes Biographies Acknowledgments Abbreviations Used References
Review
SERS-based immunoassays. The design and fabrication of SERS nanoprobes are introduced in section 2. As this part has been extensively reviewed previously,7,12−14 we briefly summarize the typical design and structure of a SERS nanoprobe. In section 3, different platforms in SERS-based immunoassay are summarized, ranging from solid substrates to liquid microspheres to microfluidic chips, etc. The advantages and disadvantages of each platform will be compared and discussed, which is expected to provide a brief guideline for choosing a suitable platform for specific applications. Aside from the nanoprobes and platforms, spectral-encoding techniques are usally employed in an immunoassay system for multiplex detection.15−17 Therefore, in section 4, the typical SERS-encoding techniques are summarized, including those based on Raman frequency or intensity, as well as several SERS-involved multimodal-encoding strategies. We then provide considerable details on the biochemical applications of SERS-based immunoassay in analyzing various targets in section 5. Finally, the challenges and future perspective of designing nextgerneration SERS immunoassays are presented in sections 6 and 7.
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1.1. Historical View of Immunoassay Protocols
An immunoassay is defined as a test for the quantitative determination of chemical substances, such as hormones, drugs, and specific proteins, that utilizes the highly specific binding between antibodies and antigens or haptens.18,19 The development of immunoassay starts from the detection of insulin by Yalow and Berson.20 They found out that the globulins bound to insulin in patients treated with exogenous animal insulin were actually antibodies. Afterward, insulin antibodies were purified and the first radio-immunoassay (RIA) was established to detect insulin using a radioactive iodine label.21 Later, together with two other scientists, Yalow won the Nobel Prize in Medicine/Physiology due to her pioneering work on devising RIA. Since its discovery, RIA has paved the way for the rapid development of immunoassays. In the late 1960s, immunoassay techniques were developed for chemically linking enzymes to antibodies. Soon after that, in 1971, the enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) were developed by Engvall and Perlmann22 and van Weeman and Schuurs,23 respectively. The replacement of radiolabels with enzymes made immunoassay much simpler and more popular. Today, most of the detection of clinical protein biomarkers is performed with ELISA kits. Since the 1970s, various detection techniques have been employed to develop the formats of immunoassays. For example, immunoassays by electrochemical techniques can determine the level of analytes by the changes in electrical signals such as potential, current, resistance, and capacitance.24 As a label-free method, electrochemical immune detection offers the advantages of fast response, low cost, and simplicity, which has a wide range of applications in biosensing. On the other hand, with the rapid development of optical materials and methods, the optical detection technique has become one of the most popular methods for immunoassays in recent years. In the beginning, chemiluminescence immunosensors have been employed for routine clinical diagnosis or biomedical research due to their advantages such as no radioactive waste, the relatively simple instrumentation, and high sensitivity. In principle, they employ chemical reactions to produce optical signals for immunoassays.48 Later, fluorescence labels, including fluorescent dyes and quantum dots, were used to develop
1. INTRODUCTION Immunoassay is one of the most famous tools for the quantitative detection of biochemical targets (e.g., proteins and toxins). The fine accuracy and operability of immunoassays have promoted thorough investigations of stringent issues, such as disease diagnosis, food safety, and environmental protection. Recent years have witnessed significant progress in developing more efficient immunoassay protocols. Immunoassays have evolved from the traditional ones performed on microplates to those conducted with highly automized platforms using various kinds of readout signals (e.g., colorimetry, electrochemistry, surface plasmon resonance, fluorescence, and surface-enhanced Raman scattering). To date, immunoassay is no longer regarded as just a simple test. It is more appropriate to consider immunoassay as a sophisticated analyte-detecting system composed of both biochemical and electrical or optical techniques. Since other immunoassay strategies have been wellreviewed elsewhere,1−3 here we only focus on optical immunoassays, especially those using surface-enhanced Raman scattering (SERS) as the readout signal. SERS is wellknown for its high sensitivity, fine specificity, and excellent multiplexing ability, leading to the blooming use of SERS-based immunoassays.4−9 The generation of Raman enhancement needs a nanoscopic metallic surface;10,11 consequently, SERSbased immunoassays have some special requirements on the layout of the assay. SERS immunoassays usually contain several basic elements (i.e., nanoprobes, SERS spectra, and assay platforms), each of which requires an elaborate design. Despite its rapid development, up to now, there is no review covering SERS immunoassay in detail. Hence, in this review, we try to comprehensively cover SERS-based immunoassays from basic principles to abundant applications. Typical structures of SERS nanoprobes, productive optical-encoding strategies, and popular immunoassay platforms are highlighted, followed by the introduction of representative biological applications using SERS immunoassays. Specifically, we start with the history, principles, and classifications of immunoassays in section 1. Then, we focus on 7911
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Chemical Reviews
Review
optical immunoassays.49,50 The advantages of fluorescence immunoassays include simplicity of system design, improved sensitivity, and fast readout process. More recently, immunoassays based on SERS, which utilize Raman reporters as the optical labels, have attracted much attention due to their ultrahigh sensitivity, excellent multiplexing ability, and high photostability.8 Aside from these labeled optical techniques, surface plasmon resonance (SPR) immunoassays provide a label-free optical sensing platform,51 in which the specific interactions between antibodies and antigens could be determined by the changes in reflective index in real time. Another kind of immunosensor employs the quartz crystal microbalance (QCM) technique, which measures the mass change through a quartz crystal resonator,52 to perform the label-free, noninvasive, and real-time detection. Summarily, the diversity of detection techniques makes it easy and flexible to design efficient immunoassays. Table 1 shows some typical examples of immunoassays using the above-mentioned techniques, in which the comparisons among different techniques are also summarized.
screening, and environmental monitoring.9 Additionally and importantly, the SERS-based optical-encoding technique has been well-employed for high-throughput detection, in which multiple targets could be distinguished simultaneously by the Raman frequency or intensity.65 Further, SERS has also been combined with fluorescence17 or spatial coordinate66 for joint encoding. The combination of SERS with other techniques greatly improved the encoding capacity, which is of great importance for achieving high-throughput analysis. In brief, SERS-based immunoassays mainly have three unique advantages. First, due to the large enhancement factor up to ∼1010, SERS-based detection could reach a high sensitivity to a single molecule level, making it extremely useful for trace analysis.67−69 Second, the narrow Raman bands could be employed for highly efficient spectroscopic encoding, which can facilitate high-throughput detection.9,70 Third, SERS is insensitive to photobleaching and quenching.71 The high stability of SERS signals allows repetitive signal measurements to reduce test errors. Owing to these advantages, SERS-based immunoassays have found an enormous number of usages in both research and practical applications.
1.2. SERS-Based Immunoassay (History, Design, and Advantages)
2. PRINCIPLES OF SERS-BASED IMMUNOASSAY Typically, the development of SERS-based immunoassay requires two critical components: the immune substrate and the SERS immunoprobe. Basically, the immune substrates are modified with targeting molecules (antibodies, aptamers, etc.) to capture the specific analytes from the samples. Afterward, the SERS probes are employed to quantify the concentration of analytes. There are two basic functions of SERS probes: (1) specifically recognizing and binding to the analytes captured by the immune substrate and (2) providing SERS signals for the quantitative detection of analytes. In accordance with the universal classification for immunoassays, generally, SERSbased immunoassays could be classified into the heterogeneous assays and homogeneous ones, noncompetitive assays and competitive ones, and labeled and label-free ones. In this section, we first introduce the basic principles of SERS-based immunoassay in different formats. Then, the design and fabrication of SERS probes are summarized. The discussion on different immune substrates (platforms) is concretely described in section 3.
Among the various detection techniques employed for immunoassays, SERS has gained increasing popularity in recent decades. In this review, we specially focus on the development of immunoassays based on SERS technique. SERS-based immunoassays refer to those that using surface-enhanced Raman scattering as the readout signal. As is well-known, Raman scattering is the inelastic scattering of a photon, which was discovered by Raman and Krishnan in liquids53 and by Landsberg and Mandelstam in crystals,54 respectively. Raman scattering can provide rich structural and quantitative information on nearly all kinds of molecules and materials. However, the weak intensity of Raman spectroscopy limits its application in biomedical and chemical studies. In 1973, Fleischmann et al. discovered that pyridine molecules adsorbed onto the rough surface of silver exhibited a dramatically enhanced Raman scattering light signal.55 The enhancement factor could reach from 103 to 1010,56−58 which has then been widely used for unltrasensitive biochemical analysis. The first typical SERS-based immunoassay was developed in 1999 using a “sandwich” scheme (Figure 1).59 In that protocol, capture antibodies are bound to a flat gold surface to form an immune substrate. Gold nanoparticles were labeled with Raman reporters and conjugated with detection antibodies to form SERS probes. Then, the target antigens could be captured by the antibodies on the substrate and subsequently be recognized by the SERS probes. The presence and concentration of a specific antigen is determined by the characteristic SERS spectrum of the Raman reporters. Importantly, the authors also demonstrated that the method could be used for multiplex detection through the SERS-encoding method. In the following decades, the platforms for SERS-based immunoassay have experienced a rapid development, ranging from solid substrates (metallic59 and nonmetallic60) to liquid phase (magnetic61 and nonmagnetic62) to microfluidic chips,63 paper devices,64 and optical fibers.28 The diversity of SERSactive platforms offers sufficient choices for analyzing proteins, disease biomarkers, ions, toxins, bacteria, viruses, cells, etc. On the other hand, there is a growing demand for multiplex, highthroughput analysis of large quantities of analytes in a single sample, especially in the fields of clinical diagnosis, drug
2.1. Construction of SERS-Based Immunoassays
According to the different separation and detection processes, SERS-based immunoassays could be classified into heterogeneous and homogeneous immunoassays (Figure 2). The heterogeneous immunoassay requires the separation of bound SERS probes from free ones and the concentration of antigens is measured through the bound SERS probes. The separation step can be fulfilled using solid substrates or liquid microbeads. On the contrary, in homogeneous SERS immunoassays, the binding between SERS nanoprobes and target molecules could induce the changes in SERS intensity, which are used for the qualitive and quantitative analysis. In general, the homogeneous immunoassay is rapid, straightforward, and more adaptable to automated systems. However, it is also more vulnerable toward nonspecific antibody−antigen cross-reactions. In contrast, heterogeneous immunoassay is excellent in detection performance, including sensitivity and reproducibility, although it requires repetitive separation procedures. When considering the binding manner of the immunocomplex, the SERS-based immunoassays can be classified into 7912
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lysozyme dimer gluten peptides CA125 β2-M ApoA1
SPR
1.7−35 nM 1.12−19.2 ng/mL
2−100 pg/mL 1 fg/mL to 1 ng/mL
AFP, GPC3 EGFR SEB NT-proBNP
SERS
1.4 nM 0.33 ng/mL 0.26 U/mL 0.55 ng/mL 7.7 ng/mL
0.1 pM 1 ng/mL cetuximab 1.3 pg/mL 0.75 fg/mL
IgG, 1.6 ng/mL; HBsAg, 1.3 ng/mL
6.7 aM
pathogen of Salmonella, Listeria, and E. coli O157 IgG/hepatitis B virus
colorimetry
yes yes
no no no no
no
no
no no no no no no no
no no no no no no
no yes
5.0 ± 0.18 pg/mL 2 ng/mL 0.93 ng/mL 2.7 × 10−5 ng/mL 1.1 × 10−5 IU/L 1.7 × 10−5 U/mL 2.0 × 10−5 ng/mL 0.01 μg/L
yes yes
0.0016 U/mL 3 pg/mL
75.0 pM 0.5 pM 14.3 pM 6.7 pM 0.03 U/mL 1.6 ng/mL 0.12 nM or 23 ng/mL in buffer, 0.18 nM or 34 ng/mL in serum
IgG, 1 ng/mL to 10 μg/mL; HBsAg, 0.34 ng/mL to 340 μg/mL
1−1000 ng/mL 1.0 × 10−3−100 ng/mL 2.0 × 10−4−20 IU/L (1.0 × 10−4)−10 U/mL (1.0 × 10−4)−10 ng/mL 0.05−1000 μg/L
yes yes
no yes
labelfree
5 ng/mL 500 pg/mL; 1 ng/mL
4.4 ± 0.1 pg/mL 0.5 ng/mL; in serum, 1 ng/mL
limit of detection
NSE CEA Cyfra21-1 SCC CA15-3 PSA EGFR
CRP AFP β-hCG CA125 CEA human prealbumin
chemiluminescence
5.0 pg/mL to 20.0 ng/mL 10−5000 ng/mL
10.0 pg/mL to 50.0 ng/mL 500 pg/mL to ca. 100 ng/mL; in serum, 1−100 ng/mL 5−500 ng/mL 500 pg/mL to ca. 10 μg/mL; 1 ng/mL to ca. 10 μg/mL 0−0.1 U/mL 10.0 pg/mL to 10.0 ng/mL
dynamic range
fluorescence
HIgG thrombomodulin
H1N1 split influenza vaccine simultaneous detection of AFP/ PSA CA 125 AFP
CEA PSA
analyte
QCM
electrochemistry
methods
Table 1. Some Typical Examples of Immunoassays Using Different Techniquesa
min min min min min
10 min 20 min 1h 1h 1h
4h 1 h
1h
1h
45 45 45 45 45
>1 h >1 h >1 h >1 h 35 min
>1 h >1 h
